Active food packaging films from alginate and date palm pit extract: Physicochemical properties, antioxidant capacity, and stability

Abstract Date palm pits are highly available and inexpensive palm date by‐products, representing a valuable source of natural antioxidants, particularly phenolic compounds. Date palm pit extract (DPPE) was prepared from these waste products and characterized for its phenolic content and in vitro antioxidant activity. Profiling DPPE by liquid chromatography coupled with mass spectrometry (LC/MS) showed the presence of dimers and trimers of (epi)catechin as the main constituents. Alginate‐based films with four increasing concentrations of DPPE (10%, 20%, 30%, and 40% w/w) were prepared by the casting method. DPPE incorporation reduced solubility values of alginate films by 37%–64% and their surface wettability by 72%–111%. The incorporation of 10% DPPE improved water vapor barrier properties and increased tensile strength (TS) and elongation at break (%E) of alginate films by more than 23%, 50%, and 45%, respectively. The film containing 40% DPPE showed the lowest loss of phenolic content (32%), DPPH (1,1‐diphenyl‐2‐picrylhydrazyl) scavenging activity (38%), and ferric reducing antioxidant power (FRAP) (30%) after storage for 3 months.

Bio-based polymers including polysaccharides, proteins, and lipids have been used to develop edible and biodegradable packaging materials which showed their efficacy in maintaining quality and prolonging the shelf life of many food products (Aloui et al., 2014;Nicolau-Lapeña et al., 2021;Paidari et al., 2021). Alginate, one of the most popular marine polysaccharides, has widely served as an interesting polymeric supporting matrix for packaging materials due to its high abundance, low cost, nontoxicity, biocompatibility, nonimmunogenicity, biodegradability, stability, and emulsifying and film-forming properties (Arroyo et al., 2020;Parreidt et al., 2018;Wang et al., 2019). Sodium alginate-based films exhibited good oxygen and grease barrier properties as well as a glossy appearance and were water-soluble, tasteless, and odorless (Gheorghita et al., 2020).
Ensuring higher stability and gradual release of active agents and maintaining critical loads for a prolonged storage time, alginate films and coatings had served as effective vehicles for antimicrobial and antioxidant agents to enhance food quality and shelf life (Parreidt et al., 2018). Several researchers have recently developed active alginate-based matrices incorporating natural antioxidants, especially polyphenols, and demonstrated their efficacy in reducing oxidation by preventing free radical generation and scavenging reactive oxygen species (Biao et al., 2019;Moreno et al., 2020;Ruan et al., 2019). Being nonvolatile compounds, polyphenols represent a good alternative for essential oils which could negatively affect food sensory attributes or may be lost by volatilization throughout storage (Vital et al., 2018;Zhang et al., 2021a). Recently, recovering phenolic compounds from cheap raw materials such as wastes and by-products from food processing industries has emerged as a promising approach to minimize the harmful impacts of waste disposal and provide renewable high-added value products (Andrade et al., 2019;Dilucia et al., 2020).
Being the earliest tree crop based on archaeobotanical data and one of the most consumed fruit crops in many arid and semiarid regions of the Middle East and North Africa (Sarah et al., 2022), date palm has been widely studied owing to its nutritional, functional, and health properties (Maqsood et al., 2020;Otify et al., 2019). However, there is relatively limited research and patents on reusing date palm by-products (Farag et al., 2021). Among the main palm date byproducts, date pits, which are produced in large quantities reaching in some countries 30% of the harvested date fruits, are mainly used as a soil amendment or animal feed (Oladzad et al., 2021). Date pits are known as a valuable source of nutrients and natural antioxidants including vitamins, phenolic compounds, tocopherols, and carotenoids (Farag et al., 2021). Accordingly, some studies have reported their use as a coffee substitute or for the extraction of date palm oil for cosmetic and pharmaceutical applications.
Recently, date pits raw products have shown good efficiency as edible-coating additives. Ahmed et al. (2020) showed that the incorporation of date pit oil at 2% in wax-coating formulations significantly improved guava fruit shelf life. Low-cost biodegradable films developed from corn starch and date pit powder revealed enhanced mechanical, antioxidant, and insulating properties (Alqahtani et al., 2021). In this context, date seeds rich in active compounds represent a suitable candidate for producing biodegradable films and coatings with increased functionality and environmental sustainability. Therefore, this study aimed at evaluating the effect of date palm pit extract (DPPE) on the physicochemical properties, antioxidant capacity, and stability of alginate films.

| Materials and reagents
In December 2019, pits from the fruits of date palm (Phoenix dactylifera L., Variety Deglet Nour) were kindly provided by a local date palm processing industry in Tozeur, a large oasis located in southwest Tunisia (Latitude of 33°55′ 10.85′′ N; Longitude of 8° 08′ 0.67′′ E). After being thoroughly washed with distilled water, palm date pits were oven-dried at 45°C for 48 h, finely ground with a Kinematica Polymix PX-MFC 90 D mill, and then stored at +4°C in amber glass jars until extraction. All solvents and reagents used in this study, sodium alginate with an average molecular weight of 80,000 Da, and glycerol as a plasticizer were purchased from Sigma-Aldrich.

| Preparation of date palm pit extract
Date palm pit extract was prepared by reflux extraction as previously described by Souissi et al. (2018) with some modifications. Briefly, 1.25 g of date pits powder was extracted under reflux with a 100 ml water-ethanol mixture (90:10) for 1 h. The obtained hydro-ethanolic extracts were filtered through a P1 glass frit (porosity 100-160 μm) and the solvent was removed under reduced pressure using an IKA rotary evaporator. Finally, DPPE was freeze-dried (Christ-Alpha) and stored in amber glass bottles at −20°C until use.

| Determination of condensed tannin content
The DPPE condensed tannin content (CTC) was carried out as described by Aires et al. (2016). After each extraction, an aliquot of 50 ml was mixed with 5 ml HCl (37%) and 10 ml formaldehyde. The mixture was left under reflux for 30 min and then filtered and washed with distilled water. The residue was placed in a drying oven at a temperature of 105 ± 3°C until the mass stabilization (MS). Stiasny's index (SI) relative to the dry mass of the extract (DW) was calculated according to the following equation:

| Determination of total polyphenolic content
The total phenolic content (TPC) of DPPE was determined by the Folin-Ciocalteu (F-C) method (Singleton & Rossi, 1965). An aliquot of DPPE (100 μl) was added to 1 ml of freshly diluted 10-fold F-C reagent. After 5 min, 2.5 ml of Na 2 CO 3 (75 g/L) was added followed by 1 h incubation in dark at room temperature. The absorbance was measured at 760 nm (JASCO V-630). Gallic acid was used as a calibration standard and TPC was expressed as mg gallic acid equivalents per gram of freeze-dried extract (mg GAE/g).

| Determination of total anthocyanin content
The determination of total anthocyanin content (TAC) was assessed by the pH-differential method based on the absorbance measurements of diluted extracts with buffer solutions at pH 1.0 and 4.5, at 520 and 700 nm (Giusti & Wrolstad, 2001). Briefly, extracts were diluted separately with 0.025 M hydrochloric acid-potassium chloride buffer (pH = 1) and 0.4 M sodium acetate buffer (pH = 4.5) until the absorbance was within a linear range. The absorbance of each solution was measured at 520 and 700 nm. The total anthocyanin content (TAC) expressed as milligrams (mg) of cyanidin-3-glucoside equivalent/g dry weight of extract (mg CGE/g DW) was calculated following the equation: where A the absorbance is (A₅₂₀-A₇₀₀) pH 1.0 -(A₅₂₀-A₇₀₀) pH 4.5 ; MW: molecular weight of cyanidin-3-glucoside (449,38 g/mol); DF: dilution factor; l: the cell path length (1 cm); ɛ: the coefficient of molecular extinction = 26.900 L mol −1 cm −1 (Giusti & Wrolstad, 2001).
Briefly, 0.9 ml of the DPPH methanol working solution (6 10 −5 M) was mixed with 0.6 ml of blank, sample, or standard (Trolox), kept in the dark for 30 min and the absorbance was recorded at 515 nm.
The radical-scavenging activity was expressed as millimole equivalent of Trolox /gram of freeze-dried extract (mmol TE/g). The determination of ferric reducing antioxidant power (FRAP) was assessed as described by Benzie and Strain (1996). A FRAP solution containing 2.5 ml (10 mM) TPTZ [(2,4,6-tri [2-pyridyl]-s-triazine)] solution in 40 mM HCl, 25 ml of 0.3 M acetate buffer (pH = 3.6), and 25 ml FeCl 3 (20 mM) was prepared. As much as 0.1 ml of DPPE was incubated at 37°C with 0.9 ml of FRAP solution for 30 min and then the absorbance was recorded at 593 nm. Results were expressed in micromole Trolox equivalents per gram (μmol TE/g) of freeze-dried extract.

| Identification of phenolic compounds by LC-MS
High-performance liquid chromatography (HPLC) analyses were carried out using a Waters Alliance system (Waters Chromatography) coupled with a photodiode array detector (PDA) and interfaced with

| Elaboration of alginate films containing DPPE
Alginate biopolymer was completely dissolved in distilled water at 70°C under mechanical stirring to obtain clear alginate filmforming solutions at 3% (w/v). DPPE was added to film-forming solutions previously cooled to 40°C to obtain the final concentrations of 0%, 10%, 20%, 30%, and 40% (w/w of alginate and DPPE). As much as 0.6 g of glycerol was added to alginate solutions under continuous agitation for 30 min. The concentrations of alginate, glycerol, and DPPE were selected based on preliminary assays where the processability, handling, and formation of homogeneous films were assured. Then, the obtained alginatebased film-forming solutions were degassed using an ultrasonic bath (BIOBASE). Twenty grams of each film-forming solution was poured into 14 cm-diameter Petri dishes and dried in a Venticell forced air convection oven (MMM group) at 40°C for 24 h to get films with an averaged thickness of 96,12 ± 0.88 μm. All films were kept in an environmental chamber at 50% RH (relative humidity) and 25°C for 1 week before testing.

| Fourier Transform Infrared analysis
The Fourier Transform Infrared (FTIR) spectra of alginate-based films were recorded between 4000 and 500 cm −1 on an FTIR Bruker spectrometer (Equinox 55, Bruker Co.) with a diamond crystal Attenuated Total internal Reflectance (ATR) accessory. A total number of 32 scans were accumulated at 4 cm −1 resolution.

| Solubility
Film samples (2 cm × 2 cm) were dried at 105°C before being weighed (W 0 ) and then soaked in 15 ml of deionized water for 24 h.
All samples were delicately wiped and dried to a constant weight (W 1 ) at 105°C. Water solubility was calculated as follows (Jouki et al., 2013):

| Surface film wettability
Film contact angle was measured in quintuplicate to determine the film's surface wettability using a Pocket goniometer PGX (Sweden) following the sessile drop method . At least six measurements on each film surface were carried out at room temperature.

| Water vapor permeability
Water vapor permeability (WVP) measurements were performed according to the ASTM standard method E96/E96M (2015). Films with an exposed area of 26.42 cm 2 were placed in permeation cells containing silica gel and stored in a controlled relative humidity (75% RH) and temperature (25°C) chamber. The WVP (g μm/m 2 d kPa) was calculated as described by Gheribi et al. (2018). Three replicates were performed for each film formulation.

| Mechanical properties
Tensile strength (TS) and elongation at break (%E) were determined on rectangular film samples (15 mm wide × 100 mm long). Eight replicates of each film formulation were tested using an Instron 3345 universal testing machine (Massachusetts, USA) following the ASTM D882-2 (2002). Measurements were performed at 23°C and 50% RH at a head speed of 20 mm min −1 .

| Color
The color parameters, lightness (L*), red-green (a*), yellow-blue (b*), and the total color difference (ΔE) were determined at least five times for each film sample using a colorimeter CM-5 (Konica Minolta). Color measurements were taken at four random points on each film.

| Light transmission and transparency
The light transmission of alginate-based films was carried out by recording their ultraviolet-visible (UV-vis) spectra at the wavelength range between 200 and 800 nm. The transparency was calculated as follows: where T600 is the transmittance at 600 nm and X is the film thickness (mm).

| Morphological characterization
The morphological analysis of the surface and cross-sections of alginate-based films was performed using a FEI Quanta 200 scanning electron microscope (FEI Company). Film samples were goldcoated using "Sputter Coater S150" under vacuum and examined using an accelerating voltage of 20 kV.

| Film stability
Total phenolic content (TPC), as well as antioxidant activity (DPPH and FRAP assays) were determined for films enriched with DPPE on days 30, 60, and 90 to test the stability of the films.

| Statistical analysis
Data were subjected to one-way analysis of variance (ANOVA) using SYSTAT (Systat software Inc.). Means comparisons were performed through 95% Fisher's least-square difference (LSD) intervals.

| Characterization of the DPPE extract
3.1.1 | Phenolic, condensed tannin, and anthocyanin contents Total phenolic (TPC), condensed tannin (CTC), and anthocyanin (TAC) contents as well as the DPPH scavenging activity and ferric reducing

| LC-MS analysis
The qualitative analysis of the phytochemicals in DPPE was per-   F I G U R E 1 LC-PDA-TIC (liquid chromatography-photodiode array detector-total ion chromatogram) profile of the date palm pit extract (DPPE). Identification of numbered peaks is given in Table 2.

| Solubility
Film solubility is intimately related to water barrier packaging performance and dictates its biodegradability behavior. Neat sodium alginate film displayed the highest water solubility (p < .05) and such high alginate hydrophilic character in high humid environments has been highlighted in the literature (Abdollahi et al., 2013;Costa et al., 2018). The incorporation of DPPE in the alginate films significantly decreased solubility values by 63% and 37% at 10% and 40% DPPE, respectively, compared with the neat alginate film (p < .05). However, no significant differences were observed between the solubility of alginate films incorporating 30% and 40% DPPE (Table 3) F I G U R E 2 Attenuated total internal reflectance-Fourier transform infrared (ATR-FTIR) spectra of the date palm pit extract (DPPE), neat alginate film (control), and alginate loaded with 10% and 40% DPPE.

F I G U R E 3 Schematic illustration of the interaction between sodium alginate (SA)
and date palm pit extract (DPPE).

| Water contact angle
Water contact angle indicates the film's surface hydrophilicity.
Neat sodium alginate film exhibited the lowest contact angle (~33°) and thus the highest hydrophilicity. DPPE incorporation significantly increased water contact angle (p < .05). The water contact angle of alginate films incorporating 10%-20% DPPE was 2 times higher than that of neat alginate film (Table 3). However, increasing DPPE content beyond 20% decreased water contact angle but this latter remained 1.7 times higher than that of alginate film. The observed increase in the water contact angle and subsequently decrease in surface wettability may be attributed F I G U R E 4 Scanning electron microscopy (SEM) micrographs of surface (×600) and cross-section (×1200) of (a) neat alginate film, (b  However, no significant differences (p > .05) were observed between the WVP of neat alginate films and those incorporating 30% and 40% DPPE (Table 3). Increasing DPPE content beyond 20% induced structural changes in the polymeric matrix, promoting the diffusion of water vapor through pinholes and bulges as revealed by SEM micrographs (Figure 4).

| Mechanical properties
Mechanical properties of packaging material, viz., mechanical strength and flexibility are correlated to its ability to resist external stresses and cracks and preserve packaging's physical and functional performances during transport and food storage. TS and %E results are shown as a function of DPPE content (Table 3).
A similar trend was observed for chitosan films after the incorporation of 0.5% (w/v) Codium tomentosum extract which by acting as a plasticizer increased chain mobility and film extensibility  (Augusto et al., 2018). An increase in %E of starch films due to the addition of fennel essential oil was also observed by Babapour et al. (2021) and explained by its softening effect, interfering with potato starch interactions.

| Optical properties
The optical properties of packaging films greatly affect product appearance and thus consumer acceptance. The optical properties of alginate and alginate/DPPE are listed in  (Table 4) tomentosum extract (0.5% w/v) and tea polyphenols (1%-5% w/w), respectively.  These results show that polyphenols from DPPE added to alginate films may act as antioxidants enhancing the packaging effectiveness in a concentration-dependent manner (Rhimi et al., 2018).

| Antioxidant capacity and stability of Alginate-DPPE films
Previous work showed a close relationship between TPC and the antioxidant activity of date pits extracts (Alqahtani et al., 2021).

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data available on request from the authors.